A variety of methods for the amplification of nucleic acids are known. For example, polymerase chain reaction (“PCR”) (see, e.g. U.S. Pat. No. 4,683,202) is a popular method for the amplification of nucleic acids. PCR methods are in vitro methods able to amplify a specific polynucleotide sequence. PCR can be used to amplify specific polynucleotide sequences, including genomic DNA, single-stranded cDNA, and mRNA among others. As described in U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,800,159 (hereby incorporated herein by reference), PCR typically comprises treating separate complementary strands of a target nucleic acid with two polynucleotide primers to form complementary primer extension products on both strands that act as templates for synthesizing copies of the desired nucleic acid sequences. By repeating the separation and synthesis steps in an automated system, essentially exponential duplication of the target sequences can be achieved.
To successfully perform a PCR reaction, the reaction must be performed at multiple different temperatures. This requires hardware or other mechanisms for repeatedly changing the temperature of the PCR reaction. In embodiments where the target nucleic acid is RNA, reverse transcription PCR (rtPCR) may be used.
Zika virus (ZIKV) is a member of the Flavivirus genus of viruses (family Flaviviridae). Other members of the genus include dengue virus (DENY), West Nile Virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitic virus (TBEV). Flaviviruses have a single-strand, positive-sense RNA genome that serves both as a genome and messenger RNA. The RNA genome is translated into a single polyprotein that is proteolytically cleaved into three structural proteins (capsid, prM, and envelope) and non-structural proteins NS1 to NS5. The virion contains a nucleocapsid composed of the capsid protein (C) and the RNA genome, surrounded by an icosahedral shell comprising both the envelope (E) glycoprotein and membrane (M) protein or the precursor membrane (prM) protein anchored in a lipid membrane.
Flaviviruses may be transmitted by the bite from an infected arthropod (e.g., mosquito or tick) and may cause widespread morbidity and mortality throughout the world. Most recently, the Zika virus has spread rapidly across the Americas following its introduction into Brazil in 2015. While Zika virus disease is usually mild with non-specific symptoms, such as fever, rash, conjunctivitis, and muscle and joint pain, there have been more severe conditions linked to the Zika virus, such as congenital microcephaly in newborns and Guillain-Barré syndrome (GBS) in adults. Thus, it has become increasingly important to be able to detect Zika virus infection in an individual.
Currently, Zika virus infection is diagnosed through detection of the viral RNA and virus isolation from blood samples, which can be time consuming. Diagnosis by serology can be difficult as the Zika virus can cross-react with other flaviviruses, such as DENY, WNV, and YFV. Thus, there is a need for better assays that are capable of specifically detecting the Zika virus. Moreover, there is a need for a rapid and simple test for detecting Zika virus infection at or near the point of service.